CN112940073A

CN112940073A - Functionalized short peptide assembly and preparation method and application thereof

Info

Publication number: CN112940073A
Application number: CN202110111668.3A
Authority: CN
Inventors: 白硕; 王胜涛; 王安河
Original assignee: Institute of Process Engineering of CAS; Nanjing Green Manufacturing Industry Innovation Research Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS; Nanjing Green Manufacturing Industry Innovation Research Institute of Process Engineering of CAS
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-06-11
Anticipated expiration: 2041-01-27
Also published as: CN112940073B

Abstract

The invention provides a functionalized short peptide assembly and a preparation method and application thereof. The preparation raw materials of the functionalized short peptide assembly comprise: fmoc dipeptide, chloroauric acid and solvent; the Fmoc dipeptide is Fmoc-YK or Fmoc-YR. The invention prepares the functional short peptide assembly by regulating the assembly behavior of Fmoc dipeptide through gold ions, takes 9-fluorenylmethyloxycarbonyl (Fmoc) modified dipeptide and chloroauric acid as raw materials, and does not introduce organic solvent or other components in the reaction process.

Description

Functionalized short peptide assembly and preparation method and application thereof

Technical Field

The invention relates to the technical fields of material science, synthesis of functionalized nano materials, regulation and control of assembly body appearance, catalysis and the like, in particular to a functionalized short peptide assembly body and a preparation method and application thereof, and particularly relates to a method for preparing a spherical functionalized short peptide assembly body by regulating Fmoc dipeptide assembly behavior through gold ions.

Background

Molecular self-assembly refers to the process by which molecules spontaneously form ordered structures under certain thermodynamic and kinetic conditions through non-covalent forces (electrostatic forces, hydrogen bonding, pi-pi stacking, and hydrophobic interactions). Inspired by functional molecule assemblies (proteins and DNA) in human bodies, a large number of polypeptides or short peptide molecules with different sequences are designed and synthesized, and then are assembled to form the polypeptide or short peptide ordered assemblies. The morphology of the assembly is influenced by a number of factors, such as: the PH, temperature, solvent, interface of the system and the introduction of other components.

Adjusting the pH of the system: (Kumaraswamy P, Lakshmann R, Sethuraman S, et al. self-assembly of Peptides: influx of substrates, pH and Medium on the formation of nanoparticles. Soft Matter,2011,7(6): 2744-.

The appearance of the assembly body is changed by changing the characteristics of the liquid-solid interface: (Wang Y, Huang R, Qi W, et al, kinetic controlled self-assembly of redox-active transferral-diphenylalanines: from nanospheres to nanofibres. nanotechnology,2013,24(46):465603.), the FF dipeptide forms a nanowire several microns long on the glass surface and assembles into a microcapsule structure on the surface of the porous membrane.

Based on the characteristic that FF and Boc-FF can be assembled into a nanotube and a nanosphere respectively, Yuran and the like researches the co-assembly behavior of FF and Boc-FF in a liquid phase (Yuran S, Razvag Y, Reches M.Coassambly of Aromatic dimers inter Biomolecular ligands Nano,2012,6(11):9559.), and by controlling the concentrations of the two and a solvent, binary co-assembly Nano-material similar to necklace (necklace) is synthesized.

According to the scheme, the short peptide assembling behavior changes by regulating and controlling the factors influencing the assembling behavior of the assembly body, so that the appearance of the assembly body changes, a series of novel assembly bodies are designed and prepared by changing the assembling environment, but the novel assembly bodies are often poor in uniformity or cannot be prepared in a large quantity, and the novel assembly bodies are often only changed in appearance and cannot obtain new functions, so that the further application of the assembly bodies is limited.

Although a series of researches for preparing a functionalized short peptide assembly by regulating the short peptide assembly behavior have been carried out, most of the researches are limited to regulating the assembly behavior of a short peptide with a specific sequence, how to apply a regulating means to a series of short peptide sequences with similar structures to prepare a series of functionalized short peptide assemblies has important guiding significance for understanding the short peptide assembly behavior and designing the functionalized assemblies.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a functionalized short peptide assembly and a preparation method and application thereof, and particularly provides a method for preparing the functionalized short peptide assembly by regulating Fmoc dipeptide assembly behavior through gold ions.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a functionalized short peptide assembly, wherein the functionalized short peptide assembly is prepared from the following raw materials: fmoc dipeptide, chloroauric acid and solvent;

the Fmoc dipeptide is Fmoc-YK or Fmoc-YR.

In the invention, after gold ions are introduced through chloroauric acid, the morphology of the assembly of Fmoc-YK and Fmoc-YR is changed from fiber to spherical particles, and the gold nanoparticles enable the spherical particles formed by assembly to show good catalytic performance, thereby realizing the functionalization of the assembly while realizing the regulation of the morphology of the assembly. Further determines the peptide sequence design principle of preparing the functionalized short peptide assembly and has certain guiding function for preparing the functionalized short peptide assembly with a specific structure.

The structural formulas of Fmoc-YK and Fmoc-YR are shown as follows:

preferably, the functionalized short peptide assembly is prepared by regulating Fmoc dipeptide assembly behavior by gold ions.

Preferably, the functionalized short peptide assembly is a nano spherical assembly.

Preferably, the particle size of the nanosphere assembly is 80-200nm, and may be, for example, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, or the like.

Preferably, the Fmoc dipeptide has a purity of 98% or more, for example, 98%, 98.5%, 99%, 99.5%, or the like (the purity content means a mass percentage content).

Preferably, the gold content in the chloroauric acid is 47.8% or more, and may be 47.8%, 47.9%, 48%, or the like, for example (the content of the pure gold means the mass percentage content).

Preferably, the solvent is water, preferably deionized water.

In a second aspect, the present invention provides a method for preparing the functionalized short peptide assembly according to the first aspect, the method comprising the following steps:

(1) dissolving Fmoc dipeptide in a solvent to obtain Fmoc dipeptide solution; dissolving chloroauric acid in a solvent to obtain a chloroauric acid solution;

(2) and (2) mixing the Fmoc dipeptide solution obtained in the step (1) with a chloroauric acid solution, and reacting to obtain the functionalized short peptide assembly.

The invention relates to a method for preparing a functionalized Fmoc dipeptide spherical assembly by regulating Fmoc dipeptide assembly behavior through gold ions, which takes 9-fluorenylmethyloxycarbonyl (Fmoc) modified dipeptide and chloroauric acid as raw materials and prepares the functionalized Fmoc dipeptide spherical assembly in deionized water. The preparation method is simple and easy to operate, the preparation is finished in an environment-friendly aqueous solution, other components are not required to be introduced in the preparation process, the prepared short peptide assemblies Au @ Fmoc-YK and Au @ Fmoc-YR are uniform in size and uniform in dispersion, and the preparation volume of the Au @ Fmoc-YR assemblies can be enlarged to 50 mL.

Preferably, in step (1), the Fmoc dipeptide solution has a concentration of 0.1 to 1mM, and may be, for example, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, or the like, preferably 0.5mM or 1 mM.

Preferably, in step (1), the concentration of the chloroauric acid solution is 5 to 20mM, and may be, for example, 5mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, etc., preferably 10 mM.

Preferably, in the step (2), the volume ratio of the Fmoc dipeptide solution to the chloroauric acid solution is (4-9):1, and may be, for example, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, and the like, and is preferably 9: 1.

Preferably, in the step (2), the reaction temperature is 4 to 60 ℃ (for example, 4 ℃,5 ℃,6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 16 ℃, 18 ℃,20 ℃, 22 ℃,24 ℃, 26 ℃, 28 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and the like), preferably 25 ℃, and the reaction time is 12 to 48h (for example, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 35h, 40h, 45h, 48h and the like), preferably 24 h.

Preferably, the preparation method of the functionalized short peptide assembly body comprises the following steps:

(1) dissolving Fmoc dipeptide with the purity of more than 98% in water to obtain 0.1-1mM Fmoc dipeptide solution; dissolving chloroauric acid in water to obtain 5-20mM chloroauric acid solution;

(2) and (2) mixing the Fmoc dipeptide solution obtained in the step (1) and the chloroauric acid solution according to the volume ratio of (4-9) to 1, and reacting at 4-60 ℃ for 12-48h to obtain the functionalized short peptide assembly.

In a third aspect, the present invention provides a use of the functionalized short peptide assembly according to the first aspect in catalyzing conversion of p-nitrophenol into p-aminophenol or catalyzing oxidation of 3,3,5, 5-tetramethylbenzidine.

In the invention, the specific steps of the functionalized short peptide assembly in catalyzing the conversion of p-nitrophenol into p-aminophenol are as follows:

(A) mixing p-nitrophenol and sodium borohydride in a solution, and standing;

(B) and (B) adding the functionalized short peptide assembly in the first aspect into the mixed solution in the step (A), and reacting to obtain p-aminophenol.

Wherein, the reaction formula for converting p-nitrophenol into p-aminophenol is as follows:

preferably, in the step (A), the molar ratio of the p-nitrophenol to the sodium borohydride is 1 (50-200), and may be, for example, 1:50, 1:60, 1:80, 1:90, 1:100, 1:120, 1:140, 1:160, 1:180, 1:200, etc.

Preferably, in step (A), the temperature of the standing is 10-30 ℃, for example, 10 ℃, 15 ℃,20 ℃, 25 ℃, 30 ℃ and the like, and the time of the standing is 5-15min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min and the like.

Preferably, in step (B), the addition amount of the functionalized short peptide assembly is 100-300. mu.L, such as 100. mu.L, 150. mu.L, 200. mu.L, 250. mu.L, 300. mu.L, etc.

Preferably, in step (B), the reaction temperature is 10-30 deg.C, such as 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, etc., and the reaction time is 2-32min, such as 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min, 30min, 32min, etc.

In the invention, the functionalized short peptide assembly of the first aspect is used for detecting the content of glutathione by catalytic oxidation of 3,3,5, 5-Tetramethylbenzidine (TMB), and the detection method specifically comprises the following steps: mixing a buffer solution, 3,5, 5-tetramethylbenzidine and a functionalized short peptide assembly, standing, adding glutathione with different concentrations, adding the functionalized short peptide assembly to enable the solution to have a characteristic absorption peak at 652nm, gradually reducing the intensity of the absorption peak at 652nm along with the addition of the glutathione, and recording the change of the absorption peak at 652nm in real time by using an ultraviolet absorption spectrum.

Preferably, the buffer solution is sodium acetate-acetic acid (NaAc-HAc) buffer (pH 3.7).

Preferably, the concentration of glutathione is 5. mu.M-1 mM, and may be, for example, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 500. mu.M, 600. mu.M, 750. mu.M, 1mM, or the like.

In the invention, the functional mechanism of the functional short peptide assembly for detecting glutathione is as follows:

oxTMB(blue)+2GSH→TMB(colorless)+GSSG+2H⁺

wherein, the functionalized short peptide assembly generates oxidized TMB by catalyzing and

oxidizing

3,3,5, 5-tetramethyl benzidine (TMB), and the solution presents blue; reduced Glutathione (GSH) is added to reduce the oxidized TMB to colorless TMB, and the reduced Glutathione (GSH) is oxidized to oxidized glutathione (GSSG). The addition of the reduced Glutathione (GSH) can cause the obvious color change of the solution, and the aim of quickly detecting the reduced glutathione is realized by colorimetry.

After adding reduced Glutathione (GSH), performing ultraviolet absorption test, and along with the change of solution color, correspondingly changing the absorbance of the solution at 652nm, and taking the change of absorption peak intensity at 652nm as a vertical coordinate and the concentration of reduced Glutathione (GSH) as a horizontal coordinate to make a standard curve, thus achieving the purpose of quantitatively detecting the concentration of glutathione in the solution within a certain concentration range (0-31 mu M).

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention prepares the functionalized short peptide assembly by regulating the assembly behavior of Fmoc dipeptide through gold ions, after the gold ions are introduced, the appearance of the assembly of Fmoc-YK and Fmoc-YR is changed from fiber into spherical particles, and the gold nanoparticles ensure that the spherical particles formed by assembly show good catalytic performance, thereby realizing the functionalization of the assembly while realizing the regulation of the appearance of the assembly;

(2) the method has the advantages of simple and easy operation, mild reaction conditions, no need of additionally adding a reducing agent, no introduction of an organic solvent or other components in the reaction process, uniform size and good dispersibility of the dipeptide nanospheres prepared by the method;

(3) the nano-particles prepared by the invention have wide application prospects in the aspects of nano-material synthesis, drug delivery and catalysis, the Fmoc-YR dipeptide nanospheres prepared by the invention are used for catalytic reduction of p-nitrophenol and catalytic oxidation research of 3,3',5,5' -tetramethyl benzidine, and the prepared dipeptide nanospheres show good catalytic performance.

Drawings

FIG. 1 is an SEM image of Fmoc-YK provided in example 1.

FIG. 2 is an SEM image of Au @ Fmoc-YK provided in example 1.

FIG. 3 is an SEM image of Au @ Fmoc-YK as provided in example 2.

FIG. 4 is an SEM image of Au @ Fmoc-YK as provided in example 3.

FIG. 5 is an SEM image of Au @ Fmoc-YK as provided in example 4.

FIG. 6 is an SEM image of Au @ Fmoc-YK as provided in example 5.

FIG. 7 is an SEM image of Au @ Fmoc-YK as provided in example 6.

FIG. 8 is an SEM image of Au @ Fmoc-YK as provided in example 7.

FIG. 9 is an SEM image of Au @ Fmoc-YR as provided in example 8.

FIG. 10 is an SEM image of Au @ Fmoc-YR as provided in example 9.

FIG. 11 is an SEM image of Au @ Fmoc-YR as provided in example 10.

FIG. 12 is a TEM image of Au @ Fmoc-YR provided in example 8, with a scale of 500 nm.

FIG. 13 is a TEM image of Au @ Fmoc-YR provided in example 8, with a scale of 100 nm.

FIG. 14 is an SEM image of Au @ Fmoc-YA as provided in comparative example 1.

FIG. 15 is an SEM image of Au @ Fmoc-YD provided in comparative example 2.

FIG. 16 is an SEM image of Au @ Fmoc-YL as provided in comparative example 3.

FIG. 17 is an SEM image of Ag @ Fmoc-YR as provided in comparative example 4.

FIG. 18 is an SEM image of Pt @ Fmoc-YR provided in comparative example 5.

FIG. 19A is a schematic diagram showing the color change of a solution during the conversion of p-nitrophenol to p-nitrophenol ions.

FIG. 19B is a schematic diagram showing the change of UV absorption during the conversion of p-nitrophenol to p-nitrophenol ions.

FIG. 20A is a schematic representation of the color change of the solution during the conversion of p-nitrophenol ions to p-aminophenol ions catalyzed by Au @ Fmoc-YR.

FIG. 20B is a schematic diagram showing the change of UV absorption during the conversion of p-nitrophenol ions into p-aminophenol ions catalyzed by Au @ Fmoc-YR

FIG. 21A is a schematic of the color change of the solution after glutathione addition.

FIG. 21B is a schematic diagram showing the change of the UV absorption peak of the solution after glutathione is added

FIG. 22 is a graph showing the change in UV absorption of solutions after the addition of glutathione at different concentrations.

FIG. 23 is a standard curve for glutathione detection using Au @ Fmoc-YR.

FIG. 24A is a schematic diagram of the color change of a solution by introducing different interference factors.

FIG. 24B is an optical schematic of UV absorption of a solution by introduction of different interference factors.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The following examples show the sources of the components as follows: Fmoc-YK (98% purity, Shanghai Ji)Erbiochemical, Inc.), Fmoc-YR (98% pure, Shanghai Gill Biochemical, Inc.), HAuCl₄(purity is analytically pure, Au is not less than 47.8%, Beijing chemical plant).

Example 1

This example provides a functionalized short peptide assembly (Au @ Fmoc-YK) prepared by the following preparation method:

(1) weighing 0.27mg of Fmoc-YK, pouring the Fmoc-YK into a 10mL plastic centrifuge tube, adding 900 mu L of multiplied by 5 deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) 900 μ L of Fmoc-YK solution was placed in a 1.5mL plastic centrifuge tube and 100 μ L of 10mM HAuCl was added₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YK nanosphere.

Wherein, FIG. 1 is SEM picture of Fmoc-YK provided in example 1, FIG. 2 is SEM picture of Au @ Fmoc-YK provided in example 1, and it can be seen from FIGS. 1 and 2 that the gold ion-mediated Fmoc-YK assembly is changed from fiber to spherical particle.

Example 2

(1) weighing 0.27mg of Fmoc-YK, pouring the Fmoc-YK into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; in thatAnd placing the mixture at 25 ℃ for reaction for 24 hours to obtain the Au @ Fmoc-YK nanospheres.

Example 3

(1) weighing 0.53mg of Fmoc-YK, pouring the Fmoc-YK into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YK nanosphere.

Wherein FIG. 2 is an SEM photograph of Au @ Fmoc-YK (0.1mM) as provided in example 1, having a size of 80nm, FIG. 3 is an SEM photograph of Au @ Fmoc-YK (0.5mM) provided in example 2, having a size of 100nm, FIG. 4 is an SEM photograph of Au @ Fmoc-YK (1mM) of 200nm size, prepared from different concentrations of Fmoc-YK and 10mM chloroauric acid, showing that the gold ion-mediated concentration of Fmoc-YK of 0.5mM is optimal for spherical shape, when the size of nanospheres is 100nm, and there is some degree of adhesion between nanospheres formed at over-diluted and over-concentrated concentrations, the reason is that the chloroauric acid and Fmoc-YK are not in the optimal matching ratio, gold ions cannot effectively induce all Fmoc-YK molecules to assemble to form nanospheres, and the yield of the nanospheres is relatively reduced due to the small amount of Fmoc-YK when the concentration is too dilute.

Example 4

(1) weighing 0.27mg of Fmoc-YK, pouring into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking by a shaker for 5min to promote sample dissolution, and performing ultrasound for 3min if the undissolved part existsCompletely dissolving the mixture to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture in a refrigerator at 4 ℃ for reaction for 24 hours, and then placing the mixture at normal temperature to obtain the Au @ Fmoc-YK nanosphere.

Example 5

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and reacting for 24 hours at 25 ℃ to obtain the Au @ Fmoc-YK nanosphere.

Example 6

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to make twoUniformly mixing the seed solution; and (3) placing the mixture in a water bath at 37 ℃ for reaction for 24h, and then placing the mixture at normal temperature to obtain the Au @ Fmoc-YK nanosphere.

Example 7

(1) weighing 0.27mg of Fmoc-YK, pouring the Fmoc-YK into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; using a pipette, 2.43mL of deionized water were pipetted to dissolve freshly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) placing the mixture in a water bath at 50 ℃ for reaction for 24h, and then placing the mixture at normal temperature to obtain the Au @ Fmoc-YK nanosphere.

FIGS. 5-8 are SEM images of Au @ Fmoc-YK at different reaction temperatures, and from a comparison of FIGS. 5-8, assembly of 0.5mM Fmoc-YK into spherical particles is best controlled by gold ions at 25 ℃. Under different temperatures, the gold ions can regulate Fmoc-YK assembly to form spherical particles, the size of the nanospheres has no obvious change, the temperature has no obvious influence on the assembly behavior, and from the aspects of convenience in operation and environmental friendliness, 25 ℃ close to normal temperature is selected as the preferred temperature.

Example 8

This example provides a functionalized short peptide assembly (Au @ Fmoc-YR) prepared by the following preparation method:

(1) weighing 0.56mg of Fmoc-YR, pouring the Fmoc-YR into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄Solutions of；

(2) To 900. mu.L of Fmoc-YR solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) reacting at 25 ℃ for 24h to obtain the Au @ Fmoc-YR nanospheres.

Fig. 12 and 13 are TEM images of Au @ Fmoc-YR provided in example 8 at different magnifications, as shown in fig. 12 and 13, the functionalized assemblies Au @ Fmoc-YR are uniform in size and uniform in dispersion, gold particles (black particles) contained in the spherical assemblies can be observed in the TEM images, and fig. 13 is a TEM image of further enlarging the assemblies, and a part of the gold particles can be observed on the surfaces of the assemblies. The existence of gold particles in the assembly lays the foundation of the catalytic performance of the assembly, and in consideration of the good dispersibility and uniformity of the assembly in the embodiment 8, the assembly in the embodiment 8 is adopted in subsequent experiments to complete related catalytic experiments.

Example 9

(1) weighing 6.8mg of Fmoc-YR, pouring the Fmoc-YR into a 15mL plastic centrifuge tube, adding 10.8mL of deionized water, shaking for 5min to promote sample dissolution, and carrying out ultrasonic treatment for 3min to completely dissolve undissolved parts so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 100. mu.L of the mother liquor to 10mL to obtain 10mM HAuCl₄A solution;

(2) to 10.8mL of Fmoc-YR solution was added 1.2mL of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) reacting at 25 ℃ for 24h to obtain the Au @ Fmoc-YR nanospheres.

Example 10

(1) 27.2mg of Fmoc-YR was weighed into a 50mL plastic centrifuge tube, 43.2mL of deionized water was added, the mixture was shaken for 5min to facilitate dissolution of the sample, and sonication was performed if any undissolved portions were presentDissolving completely for 3min to avoid insoluble substance; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 100. mu.L of the mother liquor to 10mL to obtain 10mM HAuCl₄A solution;

(2) to 43.2mL of Fmoc-YR solution was added 4.8mL of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) reacting at 25 ℃ for 24h to obtain the Au @ Fmoc-YR nanospheres.

FIGS. 9-11 are SEM images of Au @ Fmoc-YR at different reaction volumes, and it can be seen from comparison of FIGS. 9-11 that the ratio of chloroauric acid to Fmoc-YR is fixed, the reaction volume is enlarged from 1mL to 15mL or even 50mL, the morphology and size of the assembly are not affected, and the purpose of preparing a large amount of functionalized spherical assemblies (Au @ Fmoc-YR) is achieved to some extent.

Example 11

(1) weighing 0.27mg of Fmoc-YK, pouring the Fmoc-YK into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L of the mother liquor to 2mL to obtain 5mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 5mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YK nanosphere.

Example 12

(1) weighing 0.27mg of Fmoc-YK, pouring into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote sample dissolution, and performing ultrasonic treatment for 3min to completely dissolve undissolved parts so as to avoid the existence of undissolved partsDissolving; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L of the mother liquor to 0.5mL to obtain 20mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 20mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YK nanosphere.

Example 13

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) placing the mixture at 25 ℃ for reaction for 12h to obtain the Au @ Fmoc-YK nanosphere.

Example 14

(2) to 900. mu.L of Fmoc-YK solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; standing and reacting at 25 DEG CAnd (4) obtaining the Au @ Fmoc-YK nanosphere after 36 h.

Comparative example 1

The present comparative example provides a functionalized short peptide assembly (Au @ Fmoc-YA) prepared by the following preparation method:

(1) weighing 0.24mg of Fmoc-YA, pouring the Fmoc-YA into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YA solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YA assembly.

FIG. 14 is an SEM image of Au @ Fmoc-YA provided in comparative example 1, and as shown in FIG. 14, the assembly has a random morphology, which indicates that gold ions cannot effectively regulate Fmoc-YA assembly under experimental conditions to form nanospheres.

Comparative example 2

The comparative example provides a functionalized short peptide assembly (Au @ Fmoc-YD), which is prepared by the following preparation method:

(1) weighing 0.26mg of Fmoc-YD, pouring the Fmoc-YD into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) mu.L of 10mM HAuCl was added to 900. mu.L of Fmoc-YD solution₄Shaking the solution for 3min to mix the two solutions uniformly; and (3) placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YD assembly.

FIG. 15 is an SEM image of Au @ Fmoc-YD provided in comparative example 2, and as shown in FIG. 15, the assembly has a random morphology, which indicates that gold ions cannot effectively regulate the assembly of Fmoc-YD to form nanospheres under experimental conditions.

Comparative example 3

The present comparative example provides a functionalized short peptide assembly (Au @ Fmoc-YL) prepared by the following preparation method:

(1) weighing 0.26mg of Fmoc-YL, pouring into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 2.43mL of deionized water were pipetted in portions by using a pipette gun to dissolve newly purchased 1g of unpackaged HAuCl₄To obtain 1M mother liquor, diluting 10. mu.L mother liquor to 1mL to obtain 10mM HAuCl₄A solution;

(2) to 900. mu.L of Fmoc-YL solution was added 100. mu.L of 10mM HAuCl₄Shaking the solution for 3min to mix the two solutions uniformly; and placing the mixture at 25 ℃ for reaction for 24h to obtain the Au @ Fmoc-YL assembly.

FIG. 16 is an SEM image of Au @ Fmoc-YL provided in comparative example 3, and as shown in FIG. 16, the assembly has a random morphology, which indicates that gold ions cannot effectively regulate Fmoc-YL assembly to form nanospheres under experimental conditions.

Comparative example 4

This comparative example provides a functionalized short peptide assembly (Ag @ Fmoc-YR) prepared by the following preparation method:

(1) weighing 0.28mg of Fmoc-YR, pouring the Fmoc-YR into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; weigh 17mg AgNO₃Pouring into a 15mL plastic centrifuge tube, adding 10mL deionized water, shaking for 3min to promote sample dissolution to obtain 10mL 10mM AgNO₃A solution;

(2) to 900. mu.L of Fmoc-YR solution was added 100. mu.L of 10mM AgNO₃Shaking the solution for 3min to mix the two solutions uniformly; and standing at 25 ℃ for reaction for 24h to obtain the Ag @ Fmoc-YR assembly.

Figure 17 is an SEM image of Ag @ Fmoc-YR provided in comparative example 4, and as shown in figure 17, the assembly is of a random morphology, indicating that silver ions are not effective in inducing Fmoc-YR assembly to form functionalized nanospheres under experimental conditions.

Comparative example 5

This comparative example provides a functionalized short peptide assembly (Pt @ Fmoc-YR) prepared by the following preparation method:

(1) weighing 0.28mg of Fmoc-YR, pouring the Fmoc-YR into a 1.5mL plastic centrifuge tube, adding 900 mu L of deionized water, shaking for 5min to promote the dissolution of the sample, and carrying out ultrasonic treatment for 3min to completely dissolve the undissolved part so as to avoid insoluble substances; 4.83mL of deionized water are transferred in multiple times by a pipette gun to dissolve newly purchased H which is not unpacked and packaged by 1g₂PtCl₆To obtain 0.4M stock solution, diluting 100. mu.L of stock solution to 4mL to obtain 10mM H₂PtCl₆A solution;

(2) to 900. mu.L of Fmoc-YR solution was added 100. mu.L of 10mM H₂PtCl₆Shaking the solution for 3min to mix the two solutions uniformly; and standing at 25 ℃ for reaction for 24h to obtain the Pt @ Fmoc-YR assembly.

FIG. 18 is an SEM image of Pt @ Fmoc-YR provided in comparative example 5, the assembly being of a random morphology, illustrating that platinum ions are not effective in inducing Fmoc-YR assembly to form functionalized nanospheres under experimental conditions.

Application example 1

To 3mL of a 0.1mM p-nitrophenol solution was added 200. mu.L of a 200mM sodium borohydride solution, and after standing for 10min, 200. mu.L of a 0.5mM Au @ Fmoc-YR solution (wherein the 0.5mM Au @ Fmoc-YR solution means 900. mu.L of an Au @ Fmoc-YR assembly solution prepared by mixing 0.5mM Fmoc-YR with 100. mu.L of 10mM chloroauric acid) was added, and the change in the absorption peak at 400nm was recorded in real time by ultraviolet absorption spectroscopy, to design a control experiment in which 200. mu.L of double distilled water was used instead of 200. mu.L of the 0.5mM Au @ Fmoc-YR solution and the change in the absorption peak at 400nm of the solution was recorded.

Wherein, fig. 19 is a schematic diagram of ultraviolet absorption change in the process of converting p-nitrophenol into p-nitrophenol ions, and fig. 20 is a schematic diagram of ultraviolet absorption change in the process of converting the Au @ Fmoc-YR catalyzed p-nitrophenol ions into p-aminophenol ions. As shown in FIGS. 19-20, the UV absorption peak at 400nm of p-nitrophenol gradually decreased with time, and 1mmol Au @ Fmoc-YR (based on the molar amount of Fmoc-YR) could completely convert 3mmol of p-nitrophenol into p-aminophenol within 30 min.

Application example 2

To 3mL of sodium acetate-acetic acid (NaAc-HAc) buffer (pH 3.7) were added, in order, 100. mu.L of a 5mM Tetramethylbenzidine (TMB) solution and 50. mu.L of a 5mg/mL Au @ Fmoc-YR solution (5 mg/mL Au @ Fmoc-YR solution prepared by mixing 900. mu.L of 0.5mM Fmoc-YR with 100. mu.L of 10mM chloroauric acid, followed by lyophilization, weighing of the solid to prepare a 5mg/mL Au @ Fmoc-YR solution), and after leaving at room temperature for 10 minutes, 100. mu.L of Glutathione (GSH) solutions of different concentrations were added, the concentrations of glutathione being in the ranges of 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 500. mu.M, 600. mu.M, 750. mu.M and 1mM, and the addition of Fmoc, with the addition of glutathione, the intensity of the absorption peak at 652nm is gradually reduced, and the change of the absorption peak at 652nm is recorded in real time by using an ultraviolet absorption spectrum.

Wherein, FIG. 21 is a schematic diagram showing the change of the ultraviolet absorption peak of the solution after glutathione is added and the color of the solution, as shown in FIG. 21, the oxidation product generated by oxidizing TMB with Au @ Fmoc-YR is further reduced by glutathione, the absorption peak of the oxidation product at 652nm is weakened and the solution is changed from blue to colorless.

FIG. 22 is a schematic diagram showing the change of the ultraviolet absorption of the solution after glutathione is added at different concentrations. FIG. 23 is a standard curve for glutathione detection using Au @ Fmoc-YR. FIG. 24 is an optical schematic of the effect of introducing different interference factors on the UV absorption of a solution and the color change of the solution. As shown in FIGS. 22-24, the linear range of glutathione detection by Au @ Fmoc-YR is 0.15-31. mu.M, the lowest limit of detection is 0.005. mu.M, and Au @ Fmoc-YR can still specifically detect glutathione in the presence of a series of interference factors.

The applicant states that the present invention is illustrated by the above examples to show the functionalized short peptide assemblies, the preparation method and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A functionalized short peptide assembly is characterized in that the functionalized short peptide assembly is prepared from the following raw materials: fmoc dipeptide, chloroauric acid and solvent;

the Fmoc dipeptide is Fmoc-YK or Fmoc-YR.

2. The functionalized short peptide assembly of claim 1, wherein the functionalized short peptide assembly is prepared by gold ion-regulated Fmoc dipeptide assembly behavior;

preferably, the functionalized short peptide assembly is a nano spherical assembly;

preferably, the particle size of the nano spherical assembly is 80-200 nm.

3. The functionalized short peptide assembly according to claim 1 or 2, wherein the Fmoc dipeptide has a purity of 98% or more;

preferably, the content of gold in the chloroauric acid is more than 47.8%;

preferably, the solvent is water.

4. The method for preparing the functionalized short peptide assembly according to any one of claims 1 to 3, wherein the method for preparing the functionalized short peptide assembly comprises the following steps:

5. The method for preparing a functionalized short peptide assembly according to claim 4, wherein the concentration of the Fmoc dipeptide solution in step (1) is 0.1 to 1mM, preferably 0.5mM or 1 mM.

6. The method for preparing a functionalized short peptide assembly according to claim 4 or 5, wherein the concentration of the chloroauric acid solution in the step (1) is 5-20mM, preferably 10 mM.

7. The method for preparing a functionalized short peptide assembly according to any one of claims 4 to 6, wherein in the step (2), the volume ratio of the Fmoc dipeptide solution to the chloroauric acid solution is (4-9):1, preferably 9: 1.

8. The method for preparing the functionalized short peptide assembly according to any one of claims 4 to 7, wherein in the step (2), the reaction temperature is 4-60 ℃, preferably 25 ℃, and the reaction time is 12-48h, preferably 24 h.

9. The method for preparing the functionalized short peptide assembly according to any one of claims 4 to 8, wherein the method for preparing the functionalized short peptide assembly comprises the following steps:

10. Use of the functionalized short peptide assembly of any one of claims 1-3 for catalyzing the conversion of p-nitrophenol to p-aminophenol or for catalyzing the oxidation of 3,3,5, 5-tetramethylbenzidine.